Dysregulation of the immune system, which induces an aberrant activation of leukocytes, plays a critical role in the pathogenesis of a wide range of autoimmune and immunological disorders including such inflammatory bowel diseases (IBD) as ulcerativecolitis andCrohn's disease. A major challenge in treating these disorders is the lack of therapeutic strategies to manipulate specific molecular functions only in the aberrantly activated immune cells. One such strategy is to deliberately activate RNAi, a conserved gene-silencing mechanism, with an exogenous synthetic siRNA. Compared with conventional drug discovery approaches involving library searches of small-molecule chemicals, siRNAs possess an enormous advantage that can, in principle, make virtually any target molecule druggable. However, immune cells, in particular lymphocytes and monocytes, are among the most difficult targets for siRNA delivery, as they are resistant to conventional transfection reagents and disperse in the body. To address this siRNA delivery problem, our proposal aims to develop integrin-targeted stabilized nanoparticles (I-tsNP): nano-sized neutral liposomes that encapsulate siRNAs and that are directed to leukocyte subsets via a surface-attached integrin antibody or integrin ligand. Building on our preliminary data, which showed that cyclin D1 (CyD1)-siRNA, a novel anti-inflammatory drug candidate, systemically delivered with the prototype I-tsNP induced potent gene silencing and anti-inflammation in vivo, this RC1 grant application seeks to develop the capability of I-tsNP to selectively target aberrantly activated leukocytes in intestinal inflammation. Using a ligand MAdCAM-1 that preferentially binds to a high-affinity (HA) conformation of an [unreadable]4[unreadable]7 integrin, we will target inflammatory cells involved in colitis that persistently express the HA [unreadable]4[unreadable]7 integrin. The following 3 aims are proposed. Aim 1 is to test the hypothesis that surface-displayed MAdCAM- 1 can direct nanoparticles to deliver siRNAs to the HA [unreadable]4[unreadable]7 integrin-expressing cells both in vitro and in vivo. To validate the ability of MAdCAM-1-directed nanoparticles (MAdCAM I-tsNP) to deliver siRNAs in vivo, we will use a novel strain of knock-in mice that we engineered to persistently express the HA [unreadable]4[unreadable]7 integrin. Aim 2 is to validate the hypothesis that MAdCAM I-tsNP delivers siRNAs selectively to the inflamed gut. To this end, we will study the bio-distribution of siRNAs in a mouse IBD model using microPET/CT in vivo imaging. Aim 3 is to substantiate the hypothesis that CyD1-siRNA delivery with MAdCAM I-tsNP enhances therapeutic efficacy in the IBD model. The proof of concept data generated here with MAdCAM I-tsNP should help establish a highly cell-type-specific siRNA delivery platform, as well as contribute to the development of a strategy for optimizing the platform in a specific application (e.g., IBD) with microPET/CT in vivo imaging. PUBLIC HEALTH RELEVANCE: A novel class of medicines called RNAi (RNA interference) therapeutics has just emerged that harnesses the natural biological process to turn off gene expression, thereby preventing disease-causing proteins from being produced by cells. The major problem facing the realization of RNAi therapeutics is DELIVERY;i.e., how to direct intact RNAs to the right tissues in the body and then successfully escort them into cells. To find a solution to this specific problem, a team of multi-disciplinary scientists has joined together to develop a novel nanoscale delivery vehicle to target therapeutic RNAs only to disease-causing cells in such inflammatory bowel diseases (I.B.D.) as ulcerative colitis and Crohn's disease.